Guide Positioning Sequencing for DNA Methylation
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Solution Overview
Problem
Current DNA methylation detection methods, such as MethyIC-seq, face challenges in achieving accurate, high-resolution whole-genome coverage due to nucleotide imbalance, low complexity in sequencing reads, and sensitivity issues with low methylation, low GC content, and repeat sequences, limiting their ability to provide a complete map of DNA methylation in cell types or tissues.
Innovation Solution
A guide positioning sequencing technology that involves treating nucleic acid double strands with a polymerase to create 3' end deletions, followed by sodium bisulfite conversion to differentiate methylated and unmethylated cytosine, allowing for accurate methylation status determination through next-generation sequencing, with one strand used for genome positioning and the other for methylation detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sodium bisulfite conversion is used for DNA methylation detection, then accurate single base resolution detection is achieved, but nucleotide imbalance and low complexity in sequencing reads result in low mapping efficiency
Solution Approach 1:
The invention segments the sequencing read into two functional parts: one end retains the original genomic sequence for mapping to the reference genome, while the other end contains the bisulfite-converted sequence for methylation detection. This segmentation allows each part to serve its specific function optimally without interfering with the other.
Solution Approach 2:
The invention creates asymmetric treatment of DNA strands during sequencing library preparation. One strand is subjected to bisulfite conversion while the other strand serves as a template for generating the mapping sequence, creating an asymmetric workflow that preserves mapping capability while enabling methylation detection.
2Quantity of substance
If conventional methylation detection methods are used, then whole genome coverage is achieved, but sensitivity to low methylation, low GC content and repeat sequences is insufficient
Solution Approach 1:
The invention applies different quality characteristics to different parts of the sequencing data. The mapping end uses high complexity original sequence for accurate genomic positioning, while the detection end uses bisulfite-converted sequence for methylation status determination. This local quality differentiation enables sensitive detection in challenging regions.
Solution Approach 2:
The invention introduces an intermediary strategy where the unmethylated strand serves as a mediator providing the mapping sequence, while the methylated strand provides the methylation detection signal. This intermediary approach allows simultaneous achievement of whole genome coverage and high detection sensitivity.
3Quantity of substance
If MethyIC-seq is used for DNA methylation detection, then whole genome level measurement is achieved, but design defects cause strong tendency to detect only high methylation regions
Solution Approach 1:
The invention inverts the conventional approach by using the unmethylated strand (which becomes the mapping template) rather than relying solely on the methylated strand for both mapping and detection. This inversion allows the method to detect low methylation regions that would be missed by conventional approaches.
Solution Approach 2:
The invention changes the parameter of strand utilization in sequencing library preparation. Instead of using both strands equally or preferring the methylated strand, the method specifically designates one strand for mapping and the other for methylation detection, creating a parameter change that eliminates detection bias toward high methylation regions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables accurate, high-resolution DNA methylation detection at single base pair resolution, overcoming previous limitations by improving mapping efficiency and sensitivity across the genome, including regions with low GC content and repeat sequences.
Implementation Method 1
The main principle of this method is that un-methylated C (cytosine) can be converted into U (uracil) while methylated C will not change when DNA has been treated by sodium bisulfite
Implementation Method 2
Afterwards, the specific region at which sodium bisulfite has been converted into DNA is amplified via PCR
Implementation Method 3
the specific region at which sodium bisulfite has been converted into DNA is amplified via PCR
Data Source
AI summary
This invention is directed to a guide positioning sequencing technology of whole-genome DNA methylation. The invention provides a new detection method of nucleic acid methylation. In particular, a concept of “positioning” in the detection of nucleic acid methylation is provided. Specifically, a portion of a sequence is used for genome wide positioning and the other portion of the sequence is used for methylation detection in sequencing, thereby solving/defeating previously existing challenges in methylation detection and bioinformatics analysis of a genome.